Human cells express dozens of SCF ubiquitin ligases. These enzymes regulate a broad swath of cell and organismal biology by attaching ubiquitin to intracellular proteins, which often culminates in degradation of the modified protein at the hands of the 26S proteasome. Despite the importance of SCF enzymes, there is much that we still do not know about how they work. In addition, there remains much to learn about how they are regulated. One particular SCF, SCFCdc4, is the archetype for the entire cullin- RING ligase (CRL) family and studies on this and other SCF enzymes have provided a template for understanding the hundreds of CRLs expressed in human cells. Each of the dozens of different SCF complexes expressed in human cells has a distinct substrate receptor (e.g. Cdc4 in the case of SCFCdc4) which enables it to bind a unique set of substrates. The substrate receptors, known as F-box proteins (FBPs) compete for assembly with a common catalytic core composed of Cul1 and the RING domain subunit Rbx1/Roc1/Hrt1. It is thought that the FBPs are in dynamic equilibrium with the Cul1-Rbx1 module, which enables the cell to modulate its repertoire of SCF complexes to meet demand. However it is not known how or even whether this occurs. In this application, I propose to address key unanswered questions about SCF mechanism and regulation. The proposed work is divided into three specific aims. In the first aim we will use optical methods to test our hypothesis that there exists a heretofore undetected, substrate-triggered conformational change in SCF that our recent observations suggest is the rate-limiting step of the ubiquitination reaction. In the second aim, we will exploit fluorescently-tagged SCF subunits generated during the course of work on aim 1 to investigate the dynamic nature of the interaction between the substrate binding module and the catalytic core. These experiments will reveal the intrinsic properties of this interaction that must underlie any dynamic equilibrium that exists within cells. In the third aim, I propose to develop a set of novel methods based on quantitative multidimensional mass spectrometry techniques to monitor dynamics of SCF complexes within living cells. The methods developed in aim 3 will enable us to get a snapshot of the repertoire of SCF complexes in a cell at any given time and monitor how that repertoire changes over time in unperturbed cells or in response to drugs or genetic manipulations. Together, the aims proposed here will yield important new insights into how SCF complexes carry out ubiquitin conjugation and how the cellular repertoire of SCF complexes is controlled. Given that one particular CRL is the target of the important anti-cancer drug thalomid and the entire family of CRLs is targeted by a second anti-cancer drug (MLN4924) that is currently in human clinical testing, knowledge gained about the mechanism and regulation of SCF has excellent potential to be translated into development of new drugs to fight cancer. PUBLIC HEALTH RELEVANCE: Cullin-RING ubiquitin ligases (CRLs) comprise a family of enzymes that play major roles in human biology. Mutations in CRL components such as Fbw7/hCdc4 are the root cause of a significant fraction of human cancers and one particular CRL, CRL4CRBN is the target of the important anti-cancer drug thalomid. My lab co-discovered SCFCdc4, which is the archetype for all CRL enzymes. We have studied this enzyme in depth to provide a template for understanding how it and other CRLs work and how their activities are controlled. In this application I propose new investigations into how SCFCdc4 and other SCF enzymes carry out their core function of transferring ubiquitin to other proteins. I also propose experiments to investigate the mechanisms that enable cells to maintain a properly balanced repertoire of dozens of different SCF complexes. Given prior successes in developing drugs to target CRL4CRBN and the entire CRL family, the work proposed here has excellent potential to serve as a foundation for the development of new therapeutics.